This invention relates to indole derivatives useful in the treatment of a variety of diseases including restenosis, renal failure and pulmonary hypertensions, and to pharmaceutical formulations containing such compounds.
International Patent Application WO 94/14434 discloses indole derivatives which are indicated as endothelin receptor antagonists. European Patent Application 617001 discloses a large number of phenoxyphenylacetic acid derivatives which are also indicated as endothelin receptor antagonists.
Bergman et al. Tetrahedron, Vol 31, No. 17, 1975, pages 2063-2073, disclose a number of indole-3-acetic acids. Similar compounds are disclosed by Rusinova et al. Khim Geterotsikl Soedin, 1974, (2), 211-213 (see also Chemical Abstracts, Vol 81, No. 7, 19 Aug. 1974, abstract No. 37455a), and Yarovenko et al. J Gen Chem USSR (English translation), Vol 39, 1969, page 2039 (see also Beilstein, Registry Number 431619). These compounds are not indicated in any kind of therapy, and proviso (i) below relates to them.
Julian et al. J Chem Soc. Chemical Communications, No. 1, 1973, disclose an N-p-chlorobenzoylindole derivative as a by-product of a photo-addition reaction. The compound is not indicated in any kind of therapy, and proviso (ii) below relates to it.
Yamamoto et al. Japanese Patent No. 70 041 381 (see also Chemical Abstracts, Vol 75, No. 3, 1971, abstract No. 20189v), disclose an N-p-chlorobenzoylindole derivative which is indicated as an anti-inflammatory. Proviso (iii) below relates to it.
According to the present invention, there is provided a compound of formula I. 
wherein
R1 and R2 are optional substituents and independently represent C1-6 alkyl, C2-6 alkenyl [optionally substituted by CO2H or CO2(C1-6 alkyl)], C2-6 alkynyl, halogen, C1-3 perfluoroalkyl, (CH2)mAr1, (CH2)mHet1, (CH2)mCONR7R8, (CH2)mCO2R8, O(CH2)qCO2R8, (CH2)mCOR8, (CH2)mOR8, O(CH2)pOR8, (CH2)mNR7R8, CO2(CH2)qNR7R8 , (CH2)mCN, S(O)nR8, SO2NR7R8, CONH(CH2)mAr1 or CONH(CH2)mHet1;
R3 represents H, C1-6 alkyl, (CH2)pNR9R10, SO2R10, SO2NR9R10, (CH2)mCOR10, C2-6 alkenyl, C2-6 alkynyl, (CH2)mCONR9R10, (CH2)mCO2R10, (CH2)pCN, (CH2)pR10 or (CH2)pOR10;
R4 and R9 independently represent H or C1-6 alkyl;
R7 represents H, C1-6 alkyl or C1-6 alkoxy;
R5 represents H or OH;
R6 represents phenyl optionally fused to a saturated or unsaturated 5- or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from N, S, and O, the group as a whole being optionally substituted by one or more groups selected from C1-6 alkyl, C1-6 alkoxy and halogen, and wherein any members of the heterocyclic ring which are S may be substituted by one or two oxygen atoms;
R8 and R10 independently represent H, C1-6 alkyl, Ar2, Het2 or C1-6 alkyl substituted by Ar2 or Het2;
Z represents CO2H, CONH(tetrazol-5-yl), CONHSO2O(C1-4 alkyl), CO2Ar3, CO2(C1-6 alkyl), tetrazol-5-yl, CONHSO2Ar3, CONHSO2 (CH2)qAr3 or CONHSO2(C1-6alkyl);
m represents 0, 1, 2 or 3;
n represents 0, 1 or 2;
p represents 2, 3 or 4;
q represents 1, 2 or 3;
Ar1-3 independently represent phenyl, naphthyl, or an aromatic heterocycle having 5 or 6 ring members up to 4 of which are selected from N, S, and O, which aromatic heterocycle is optionally fused to a benzene ring, and which phenyl group is optionally fused to an aromatic heterocycle as defined immediately above, the group as a whole being optionally substituted by one or more groups falling within the definition of R1 above; and
Het1 and Het2 independently represent a non-aromatic heterocycle having 5 or 6 ring members up to 4 of which are selected from N, S, and O, which group is optionally substituted by one or more groups falling within the definition of R1 above, and is further optionally substituted by xe2x95x90O or xe2x95x90S; provided that:
(i) when R1 represents methoxy or is absent, R2 is absent, R3 represents H, R4 represents H, methyl or ethyl, and R6 represents unsubstituted phenyl, then Z does not represent CO2H or CO2(C1-6 alkyl);
(ii) when R1 and R2 are absent, R3 represents CO(p-ClC6H4), R4 represents H, and R6 represents unsubstituted phenyl, then Z does not represent CO2(C1-6 alkyl); and
(iii) when R1 represents methoxy, R2 is absent, R3 represents CO(p-ClC6H4), R4 represents methyl, and R6 represents unsubstituted phenyl, then Z does not represent CO2H;
or a pharmaceutically acceptable derivative thereof.
Pharmaceutically acceptable derivatives include those compounds in which the functional groups explicitly recited above have been derivatised to provide prodrugs which can be converted to the parent compound in vivo. Such prodrugs are discussed in Drugs of Today, Vol 19, 499-538 (1983) and Annual Reports in Medicinal Chemistry, Vol 10, Ch 31 p306-326. In addition, pharmaceutically acceptable derivatives include pharmaceutically acceptable salts, such as alkali metal salts (for example sodium salts) of any acidic groups that may be present.
xe2x80x9cHalogenxe2x80x9d includes fluorine, chlorine, bromine and iodine.
Alkyl groups which R1-4, R6-10 and Z represent or comprise may be straight chain, branched or cyclic.
Besides phenyl and naphthyl, specific groups that Ar1-3 may represent or comprise include indolyl, pyridinyl, thienyl, oxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, thiazolinidyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl and pyrimidinyl.
Specific groups that Het1 and Het2 may represent or comprise include oxazolidinl, triazolethione, triazolone, oxadiazolone, oxadiazolethione, imidazolidinyl, morpholinyl, piperidinyl and piperazinyl.
Preferred groups of compounds which may be mentioned include those in which:
(a) R1 represents haloen, (CH2)mCONR7R8, (CH2)mCO2R8, (CH2)mCOR8, (CH2)mOR8 or (CH2)mCN. In these groups it is preferred that R7 and R8 represent H or C1-6 alkyl. Preferably, m is 0 or 1. Thus, specific groups which may be mentioned are CONH2, CO2H, CH2OH, F or CH3CO. R1 is preferably attached to the 6-position of the indole ring.
(b) R2 is absent (i.e. its place on the indole ring is occupied by H).
(c) R3 represents H, C1-6 alkyl or (CH2)pOR10. Preferably, R10 is C1-6 alkyl and p is 2.
Thus, specific groups which may be mentioned are methyl and (CH2)2OCH3.
(d) R4 represents H.
(e) R5 represents H.
(f) R6 represents phenyl fused to a saturated 5-membered heterocyclic ring, for example 3,4-methylenedioxyphenyl.
(g) Z represents CO2H or CONHSO2Ar3. Preferably, Ar3 is phenyl substituted by one or more groups selected from C1-6 alkyl, C1-6 alkoxy and C1-6 alkyl substituted by carboxy.
Thus, specific groups which may be mentioned are: 
There is further provided a process for the production of the compounds of the invention, comprising:
(a) when R5 represents H, reaction of a compound of formula IIA. 
xe2x80x83wherein R1-4 are as defined above, with a compound of formula III. 
xe2x80x83wherein R6 and Z are as defined above, in the presence of a Lewis acid or trifluoroacetic acid, and a tri(C1-6 alkyl)silane:
(b) when R5 represents OH, reaction of a compound of formula IIA, as defined above, with a compound of formula III, as defined above, in the presence of a Lewis acid;
(c) when R3 represents H and R5 represents H, treatment of a compound of formula IIB, 
xe2x80x83wherein R1, R2 and R4 are as defined above, with a Grignard reagent, followed by reaction with a compound of formula III, as defined above, followed by treatment with a Lewis acid or trifluoroacetic acid, and a tri(C1-6 alkyl)silane:
(d) when R3 represents H and R5 represents H, treatment of a compound of formula IIB, as defined above, with a Grienard reagent, followed by reaction with a compound of formula IV, 
xe2x80x83wherein R6 and Z are as defined above, and Hal represents halogen:
(e) when R5 represents H, reaction of a compound of formula IIA, as defined above, with a compound of formula IV, as defined above, in the presence of a hindered, non-nucleophilic base;
(f) reacting a compound of formula I, in which R1 represents Br, with CO gas in the presence of a palladium catalyst and a reducing agent, to provide the corresponding compound of formula I in which R1 represents CHO;
(g) reacting a compound of formula I, in which R1 represents Br, with CO gas in the presence of a palladium catalyst and a C1-6 alkanol, to provide the corresponding compound of formula I in which R1 represents CO2(C1-6 alkyl);
(h) coupling, a compound of formula I in which Z represents CO2H with a compound of formula VI.
xe2x80x83H2NSO2Ar3xe2x80x83xe2x80x83VI
xe2x80x83Wherein Ar3 is as defined above, to provide the corresponding compound of formula I in Which Z represents CONHSO2Ar3; or
(i) reacting a compound of formula I, in which R1 represents Br, with an alkyl lithium reagent and quenching with dimethylformamide or carbon dioxide, to give a corresponding compound in which R1 represents CHO or CO2H respectively;
and where desired or necessary converting the resulting compound of formula I into a pharmaceutically acceptable derivative thereof or vice versa.
In process (a), suitable Lewis acids include boron trifluoride diethyletherate. The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example dichloromethane, at a temperature below room temperature, for example xe2x88x9240 to xe2x88x9278xc2x0 C. A preferred tri(C1-6 alkyl)silane is triethylsilane. Intermediate compounds in which R5 represents OH may be isolated from this process.
In process (b), suitable Lewis acids include boron trifluoride diethyletherate. The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example dichloromethane, at a temperature below room temperature, for example xe2x88x9240 to xe2x88x9278xc2x0 C. The reaction is followed by basic work up.
In process (c), suitable Grignard reagents include methylmagnesium iodide. The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example toluene, below room temperature, for example xe2x88x9270xc2x0 C. Suitable Lewis acids include boron trifluoride diethyletherate. The acid treatment may be carried out in a solvent which does not adversely affect the reaction, for example dichloromethane, at a temperature of 0xc2x0 C. to room temperature. A preferred tri(C1-6 alkyl)silane is triethylsilane.
In process (d), suitable Grignard reagents include methylmagesium iodide. The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example toluene, at or around room temperature. The reaction mixture may be worked up with a weak acid such as aqueous ammonium chloride. Hal is preferably Br.
In process (e), suitable hindered non-nucleophilic bases include 2,6-dimethylpyridine. The reaction is preferably carried out in a solvent which does not adversely affect the reaction. for example dimethylformamide, at an elevated temperature, for example 80xc2x0 C.
In process (f), suitable palladium catalysts include dichlorobis(triphenylphosphine)-palladium(II). Suitable reducing agents include sodium formate. The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example dimethylformamide, at an elevated temperature, for example 110xc2x0 C.
In process (g), suitable palladium catalysts include dichlorobis(triphenylphosphine)-palladium(II). The reaction is preferably carried out in a solvent which does not adversely affect the reaction, for example dimethylformamide, at an elevated temperature, for example the reflux temperature of the reaction mixture.
In process (h), the reaction may be facilitated by the use of conventional coupling agents, for example N,N-carbonyl diimidazole. When using this agent, the acid is first reacted with the agent (for example in dichloromethane at the reflux temperature of the solvent), and then the product of this reaction is reacted with the amine (preferably in the presence of a strong hindered amine base such as 1,8-diazabicyclo[5.4.0]undec-7-ene, in a solvent such as dichloromethane at the reflux temperature of the solvent). An alternative agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide which reacts at room temperature.
In process (i), suitable alkyl lithium reagents include n-butyl lithium. The reaction is carried out by adding the alkyl lithium reagent to the compound of formula I in a solvent such as tetrahydrofuran, at a temperature below room temperature (for example xe2x88x9240 to xe2x88x9278xc2x0 C.), and stirring for about 2 hours. Dimethylformamide or solid carbon dioxide is then added and the reaction mixture allowed to warm to room temperature.
Compounds of formulae IIA, IIB, III, IV and VI are either known or may be prepared by conventional methods well known to those skilled in the art. For example, compounds of formulae IIA and IIB may be prepared by the Fischer, Reissert and Madelung syntheses. In addition, International Patent Application WO 94/14434 discloses a number of routes to 2-carboxy indole derivatives (see page 8 onwards) which may be decarboxylated readily (using copper and quinoline) to give compounds of formulae IIA or IIB in which R4 is H, or reduced to give compounds of formulae IIA or IIB in which R4 is alkyl. Other methods for the preparation of indoles are described by Moyer et at. J Org Chem, 1986, 51, 5106-5110; Wender et al. Tetrahedron, 1983, 39 No. 22, 3767-3776; Uhle, J Am Chem Soc, 1949, 71, 761: Uhle et al. J Am Chem Soc, 1960, 82, 1200; Nagasaka et al, Heterocycles, 1977, 8, 371; Bowman et al. J Chem Soc, Perkin Trans 1, 1972, 1121; Bowman et al. J Chem Soc, Perkin Trans 1, 1972, 1926; and Clark et al, Heterocycles, 1984, 22, 195.
Compounds of formula III in which R6 is an electron-rich group (for example 1,3-benzodioxole) and Z is CO2CH2CH3 may be prepared by a Friedel-Crafts acylation between a compound of formula R6H and the compound of formula ClCOCO2CH2CH3. The reaction is preferably carried out in the presence of a Lewis acid (for example AlCl3). in a solvent which does not adversely affect the reaction, for example dichloromethane, below room temperature (for example 0xc2x0 C.).
Compounds of formula III in which R6 is not an electron-rich group (for example groups substituted by halogen or OH) and Z is CO2CH3 may be prepared by reaction of a compound of formula R6Li with a compound of formula CH3OCOCO2CH3. The reaction may be carried out in a solvent which does not adversely affect the reaction, for example tetrahydrofuran, below room temperature (for example xe2x88x9240xc2x0 C. to xe2x88x9278xc2x0 C.).
Compounds of formula R6Li may be prepared by reacting a compound of formula R6Br and butyl lithium. The reaction may be carried out in a solvent which does not adversely affect the reaction, for example tetrahydrofuran, below room temperature (for example xe2x88x9278xc2x0 C.).
Compounds of formula IV may be prepared by halogenating the corresponding alcohol with an agent such as hydrobromic acid. When Z represents CO2(C1-6 alkyl), compounds of formula R6CH(OH)Z may be prepared by reacting an aldehyde of formula R6CHO with bromoform under basic conditions, and treating the crude carboxylic acid intermediate with a C1-6 alkanol.
Compounds of formula I may be converted into other compounds of formula I using known techniques. Processes (f)-(i) above are such conversions of particular interest.
Compounds of formulae I, III or IV in which Z represents a carboxylic ester may be converted into corresponding compounds in which Z represents other groups by conventional methods.
Compounds of formulae I, IIA or IIB in which R3 represents H may be converted to corresponding compounds in which R3 is other than H by conventional methods. In general. R3 groups other than H may be added by treatment of a compound of formula I, IIA or IIB in which R3 represents H with sodium hydride, followed by an appropriate compound of formula R3Br or R3I, in dimethylformamide at 0xc2x0 C. Preferably, compounds of formulae I, IIA or IIB in which R3 represents electron-withdrawing groups (such as SO2R10, SO2NR9R10, CONR9R10 and COR10) are prepared by reacting a compound of formula I, IIA or IIB in which R3 represents H with an appropriate compound of formula R3Cl.
The compounds of the invention may be separated and purified by conventional methods.
It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example as described in xe2x80x98Protective Groups in Organic Synthesisxe2x80x99 by T W Greene and P G M Wuts, John Wiley and Sons Inc, 1991. For example, it may be desirable to protect the indole nitrogen of a compound of formula IIA and use the method of process (a) followed by deprotection to give a compound of formula I in which R3 represents H. Processes (a)-(h) embrace such protection and deprotection steps.
The synthesis of triazolethione, oxadiazoione and oxadiazolethione is described in J Med Chem, 1993, 36, 1090-1099. The synthesis of oxathiadiazoie is described in Bioorganic and Medicinal Chemistry Letters, 1994, 4 No. 1, 41-44.
The compounds of the invention may possess one or more chiral centres and so exist in a number of stereoisomeric forms. All stereoisomers and mixtures thereof are included in the scope of the present invention. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of formula I.
The compounds of the invention are useful because they have pharmacological activity in animals, including humans. More particularly, they are useful in the treatment of restenosis, renal failure, pulmonary hypertension, benign prostatic hypertrophy, congestive heart failure, stroke, angina, atherosclerosis, cerebral and cardiac ischaemia and cyclosporin induced nephrotoxicity. The treatment of restenosis, renal failure and pulmonary hypertension are of particular interest. The compounds of the invention may be administered alone or as part of a combination therapy.
Thus, according to a further aspect of the invention, there is provided a compound of formula I, as defined above, but without provisos (i) and (ii), or a pharmaceutically acceptable derivative thereof, for use as a pharmaceutical.
There is further provided a pharmaceutical formulation comprising a compound of formula I, as defined above, but without provisos (i) and (ii), or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable adjuvant, diluent or carrier.
The invention also provides the use of a compound of formula I, as defined above, but without provisos (i)-(iii), or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for the treatment of restenosis, renal failure, pulmonary hypertension, benign prostatic hypertrophy, congestive heart failure, stroke, angina, atherosclerosis, cerebral and cardiac ischaemia or cyclosporin induced nephrotoxicity. The invention also provides a method of treatment of these diseases, which comprises administering a therapeutically effective amount of a compound of formula I, as defined above, but without provisos (i)-(iii), or a pharmaceutically acceptable derivative thereof, to a patient in need of such treatment.
Without being limited by theory, the compounds of the invention are believed to be endothelin receptor antagonists. Endothelin (ET) is a potent vasoconstrictor synthesised and released by endothelial cells. There are three distinct isoforms of ET: ET-1, ET-2 and ET-3, all being 21-amino acid peptides and herein the term xe2x80x98endothelinxe2x80x99 refers to any or all of the isoforms. Two receptor subtypes, ETA and ETB have been pharmacologically defined (see for example H, Arai et al, Nature, 348, 730, 1990) and further subtypes have recently been reported. Stimulation of ETA promotes vasoconstriction and stimulation of ETB receptors causes either vasodilation or vasoconstriction.
The effects of endothelin are often long-lasting and, as the endothelins are widely distributed in mammalian tissues, a wide range of biological responses have been observed in both vascular and non-vascular tissue. The main effects of endothelin are observed in the cardiovascular system, particularly in the coronary, renal, cerebral and mesenteric circulation.
Increased circulating levels of endothelin have been observed in patients who have undergone percutaneous transluminal coronary angioplasty (PTCA) (A. Tahara et al. Metab. Clin. Exp. 40, 1235, 1991) and ET-1 has been found to induce neointimal formation in rats after balloon angioplasty (S. Douglas et al. J. Cardiovasc. Pharm., 22 (Suppl 8), 371, 1993). The same workers have found that an endothelin antagonist, SB-209670, causes a 50% reduction in neointimal formation relative to control animals (S. Douglas et al. Circ Res, 75, 1994). Antagonists of the endothelin receptor may thus be useful in preventing restenosis post PTCA.
Endothelin-1 is produced in the human prostate gland and endothelin receptors have been identified in this tissue. Since endothelin is a contractile and prolifrative agent endothelin antagonists could be useful in the treatment of benign prostate hypertophy.
There is widespread localisation of endothelin and its receptors in the central nervous system and cerebrovascular system (R. K. Nikolov et al. Drugs of Today, 28(5), 303, 1992) with ET being implicated in cerebral vasospasm, cerebral infarcts and neuronal death. Elevated levels of endothelin have also been observed in patients with:
Chronic renal failure (F. Stockenhuber et al. Clin Sci (Lond.), 82, 255, 1992)
Ischaemic Heart Disease (M. Yasuda Am. Heart J., 119, 801, 1990)
Stable or unstable angina (J. T. Stewart. Br. Heart J. 66, 7 1991)
Pulmonary Hypertension (D. J. Stewart et al. Ann. Internal Medicine, 114, 464, 1991)
Congestive heart failure (R. J. Rodeheffer et al. Am.J.Hypertension, 4, 9A, 1991)
Preeclampsia (B. A. Clark et al. Am.J.Obstet.Gynecol., 166, 962, 1992)
Diabetes (A. Collier et al. Diabetes Care, 15 (8), 1038, 1992)
Crohn""s disease (S. H. Murch et al. Lancet, 339, 381, 1992)
Atherosclerosis (A. Lerman et al. New Eng. J. Med., 325, 997, 1991)
In every case the disease state associated with the physioloaically elevated levels of endothelin is potentially treatable with an endothelin receptor antagonist and hence a compound of the invention.
Compounds that selectively antagonise the ETA receptor rather than the ETB receptor are preferred.
The biological activity of the compounds of the invention may be demonstrated in Tests A-C below:
A. Binding Assay
Competition between test compounds and 125I-ET-1 binding to human endothelin receptors is determined as follows.
Binding to ETA Receptors
25 ul of a 30 pM solution of [125I]Tyr13 ET-1 (specific activity 2,200 Ci/mM) is mixed with 25 xcexcl samples of test compound (final concentrations in the range 0.1 nM-50.000 nM). 200 xcexcl of a solution containing cloned human ETA receptor (0.75 pmoles receptor protein/ml). 50 mM Tris, 0.5 mM CaCl2, 0.1% human serum albumen. 0.1% bacitracin. 0.05% Tween 20, pH 7.4 is added. The solution is mixed at 37xc2x0 C. for 2 hours. After the incubation, the unbound ligand is separated from receptor bound ligand by filtration with a Brandel cell harvester, followed by three washes of buffer. Filter papers are counted for radioactivity, and the IC50 (the concentration of test compound at which 50% of the radio-labelled compound is unbound) determined for the concentration range tested.
Binding to ETB Receptors
25 xcexcl of a 30 pM solution of [125I]Tyr13 ET-1 (specific activity, 2.200 Ci/mM) is mixed with 25 xcexcl samples of test compound (final concentration 0.1 nM-50.000 nM), 200 xcexcl of a solution containing cloned human ETB receptor (0.25 pmoles receptor protein/ml), 50 mM Tris, 0.5 mM CaCl2, 0.1% human serum albumen, 0.1% bacitracin, 0.05% Tween 20, pH 7.4 is added. The solution is mixed at 37xc2x0 C. for 2 hours. After the incubation, the unbound ligand is separated from receptor bound ligand by filtration with a Brandel cell harvester, followed by three washes of buffer. Filter papers are counted for radio-activity, and the IC50 (the concentration of test compound at which 50% of the radio-labelled compound is unbound) determined for the concentration range tested.
B. In vitro Vascular Smooth Muscle Activity
Rat Aorta
Rat aortae are cleaned of connective tissue and fat and cut into helical strips approx 4 mm in width. The endothelium is removed by dragging the luminal surface of the tissue gently across filter paper moistened with Krebs solution of composition (mM) NaCl 130, KCl 5.6, NaHCO3 25, Glucose 11.1, NaH2PO4 0.6, CaCl2 2.16, MgCl2 0.5, gassed with 95% O2/5% CO2. The strips are mounted in isolated organ baths in Krebs solution under a resting tension of 1 gram. Organ bath solutions are maintained at 37xc2x0 C. and continuously aerated with 95% O2/5% CO2. Tensions are measured with Maywood Industries isometric force transducers and displayed on Gould TA4000 recorders. After equilibration in the organ bath for 1 hour, tissues are contracted by the addition of KCl to a final concentration of 60 mM. The KCl is removed by replacing the Krebs solution, with two further washes with Krebs solution. To determine the potency of an ETA receptor antagonist, two tissues are cumulatively dosed with ET-1 (0.1 nM-1 xcexcM); other tissues are dosed with ET-1 (0.1 nM-1 xcexcM) in duplicate, beginning 30 minutes after the inclusion in the organ bath medium of the test compound. Sufficient tissues are used per experiment to generate dose-response curves to ET-1 in the absence and the presence of at least 3 concentrations of antagonist. Data are expressed as the meanxc2x1s.e.m. Dissociation constants (kb) of competitive antagonists are calculated by the method of Arunlakshana and Schild.
Rabbit Pulmonary Artery
Isolated rabbit pulmonary arteries are cleaned of connective tissue and fat and cut into rings approx 4 mm in width. The endothelium is removed by inserting a fibrous instrument moistened with Krebs solution of composition (mM) NaCl 130, KCl 5.6, NaHCO3 25. Glucose 11.1, NaH2PO4 0.6, CaCl2 2.16, MgCl2 0.5, gassed with 95% O2/5% CO2. The rings are mounted in isolated organ baths in Krebs solution under a resting tension of 1 gram. Organ bath solutions are maintained at 37xc2x0 C. and continuously aerated with 95% O2/5% CO2. Tensions are measured with Maywood Industries isometric force transducers and displayed on Gould TA4000 recorders. After equilibration in the organ bath for 1 hour. tissues are contracted by the addition of KCl to a final concentration of 60 mM. The KCl is removed by replacing the Krebs solution, with two further washes with Krebs solution. To determine the potency of an ETB receptor antagonists, two tissues are cumulatively treated with BQ-3020 (0.1 nM-1 xcexcM); other tissues are treated with BQ-3020 (0.1 nM-1 xcexcM) in duplicate, beginning 30 minutes after the inclusion in the organ bath medium of the test compound. Sufficient tissues are used per experiment to generate dose-response curves to BQ-3020 in the absence and the presence of at least 3 concentrations of antagonist. Data are expressed as the meanxc2x1s.e.m. Dissociation constants (kb) of competitive antagonists are calculated by the method of Arunlakshana and Schild.
C. In vivo Blockade of Endothelin-induced Blood Pressure Elevation
In anaesthetised, ganglion-blocked and artificially respired rats, the left common carotid artery and the right jugular vein are cannulated for the measurement of arterial blood pressure and the administration of compound respectively. Rats are treated with the ETB antagonist BQ-788 (0.25 mg/kg i.v.). Beginning 10 minutes after administering BQ-788, the hypertensive response to ET-1 (1 xcexcg/kg i.v.) is determined. When the blood pressure has returned to baseline, the test compound is administered (0.1-20 mg/kg i.v.) and after 10 minutes the ET-1 challenge is repeated. Increasing concentrations of the test compound are administered, followed 10 minutes after each administration by a further ET-1 challenge. An IC50 is determined based upon inhibition of ET-1 induced pressor response upon cumulative dosing with compound.
Duration of blockade is determined in anaesthetised, ganglion-blocked and artificially respired rats, in which the left common carotid artery and the right jugular vein are cannulated for the measurement of arterial blood pressure and the administration of compound respectively. Rats are treated with the ETB antagonist BQ-788 (0.25 mg/kg i.v.). Beginning 10 minutes after administering BQ-788, the hypertensive response to ET-1 (1 xcexcg/kg i.v.) is determined. When the blood pressure has returned to baseline, the test compound is administered (10 mg/kg i.v.). Further administrations of ET-1 are made 5, 20 and 60 minutes after dosing the test compound. In separate animals, prepared similarly, an ET-1 challenge is made 2 or 4 hours after dosing with the test compound, in these animals BQ-788 is dosed 10 minutes before the ET-1 challenge. For later time points, rats are dosed with the test compound (10 mg/kg) i.v. via a tail vein or p.o., they are then anaesthetised and prepared for blood pressure measurement as above. In these rats, ET-1 (1 xcexcg/kg i.v.) was administered 6 or 8 hours after the test compound.
For human use the compounds of the invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example they can be administered orally in the form of tablets containing such excipients as starch or lactose or in capsules or ovules either alone or in admixture with excipients or in the form of elixirs, solutions or suspensions containing the compound or salt in a liquid carrier, for example a vegetable oil, glycerine or water with a flavouring or colouring agent. They can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parental administration, they are best used as sterile aqueous solutions which may contain other substances, for example, enough glucose or salts to make the solution isotonic with blood. For parenteral administration the compound or salt may also be administered as a solution or suspension in a suitable oil, for example polyethylene glycol, lecithin or sesame oil.
Compounds of the invention may also be administered through inhalation of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane.
For oral or parenteral administration to human patients the daily dosage levels of compounds of the invention will be from 0.01 to 30 mg/kg (in single or divided doses) and preferably will be in the range 0.01 to 5 mg/kg. Thus tablets will contain 1 mg to 0.4 g of compound for administration singly or two or more at a time, as appropriate. The above dosages are, of course only exemplary of the average case and there may be instances where higher or lower doses are merited, and such are within the scope of the invention.
Alternatively the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder or in the form of a medicated plaster, patch or membrane. For example they may be incorporated in a cream containing an aqueous emulsion of polyethylene glycols or liquid paraffin. The compounds may also be administered intranasally.